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PubMed1999

Binding of extracellular matrix proteins by animal strains of staphylococcal species.

All 81 strains of Staphylococcus species isolated mainly from animals express high surface hydrophobicity as a stable property upon cultivation on blood agar. Bovine lactoferrin, human vitronectin, human fibronectin, heparin, human and bovine serum albumin were immobilized on latex beads to detect protein-binding cell surface components of 67 non-autoaggregating staphylococcal strains by a particle agglutination assay. Bovine lactoferrin was bound well by 22 strains (3 or 2) while 15 strains reacted weakly (1) and 30 did not react (0) with the lactoferrin-coated latex beads. The particle agglutination assay showed similar differences among staphylococcal strains in binding other proteins with the exception of human and bovine serum albumins for which 66 of 67 strains were negative (0). The specificity of the agglutination reaction was confirmed by a particle agglutination inhibition assay by preincubating bacterial cells with the protein (lactoferrin, vitronectin, etc.) used subsequently in particle agglutination assay. Autoaggregating strains together with some non-autoaggregating strains were selected for microtitre plate assay. According to absorbance at 570 nm, 14 strains were classified as non-adherent, 16 strains as weakly adherent and 18 strains as strongly adherent to bovine lactoferrin in microtitre plate assays. A direct correlation was found between the absorbance values at 570 nm of microtitre plate binding assay and test values obtained in particle agglutination assay. Binding of bovine lactoferrin to 81 staphylococcal strains as well as of human vitronectin and human fibronectin to a selected number of these strains was studied with radiolabeled (125I-labeled) proteins. Radiolabeled bovine lactoferrin was bound common by all except four strains (7 to 39%). Staphylococcal strains isolated from diseased pigs commonly bound 125I-labeled vitronectin (21 to 42% of the vitronectin added). Binding of vitronectin and lactoferrin was efficiently inhibited by preincubating of staphylococcal cells with sulphated carbohydrate compounds as heparin, dextran sulphate and fucoidan, but not by other non-sulphated highly charged glycoconjugates such as hyaluronic acid.

Animals↗
PubMed1996

Monocyte and neutrophil adhesion to matrix proteins is selectively enhanced in the presence of inflammatory mediators.

The authors investigated the time course of monocyte and neutrophil adhesion to fibronectin, vitronectin and albumin precoated culture wells, using mixed leucocyte populations from healthy blood donors. Moreover, the influence of chemotactic agonists on the adhesion properties as well as the quantitative expression of CD29, CD11b/CD18 and CD61 was analysed by flow cytometry. Different chemotactic agonists were used representing a classical chemotactic agonist (fMLP), and agonists with a preferential effect on monocytes (RANTES) and neutrophils (IL-8), respectively. The authors found a gradual increase in monocyte and neutrophil adhesion to all three surfaces, reaching a plateau at 15 min of incubation. Adhesion to fibronectin was significantly higher at all time points (5, 15 and 60 min, respectively) compared with vitronectin and albumin in both monocytes and neutrophils. Neutrophil adhesion to vitronectin was significantly lower at 60 min compared with 15 min. Monocyte adhesion to albumin was increased by fMLP and RANTES and to vitronectin also by IL-8. Neutrophil adhesion to albumin and vitronectin was increased by fMLP and IL-8, but not RANTES. The adhesion to fibronectin was not altered by any of the chemotactic agonists used. The quantitative levels of CD11b/CD18, but not CD29 and CD61, was increased by fMLP, but not RANTES nor IL-8. The authors conclude that the adhesion of human monocytes and neutrophils to vitronectin and albumin, but not fibronectin, is selectively enhanced by chemotactic agonists and may contribute to the selective accumulation of different leucocyte subsets at the inflammatory site.

Adolescent↗
PubMed1996

Nectinepsin: a new extracellular matrix protein of the pexin family. Characterization of a novel cDNA encoding a protein with an RGD cell binding motif.

We report the isolation and characterization of a novel cDNA from quail neuroretina encoding a putative protein named nectinepsin. The nectinepsin cDNA identifies a major 2.2-kilobase mRNA that is detected from ED 5 in neuroretina and is increasingly abundant during embryonic development. A nectinepsin mRNA is also found in quail liver, brain, and intestine and in mouse retina. The deduced nectinepsin amino acid sequence contains the RGD cell binding motif of integrin ligands. Furthermore, nectinepsin shares substantial homologies with vitronectin and structural protein similarities with most of the matricial metalloproteases. However, the presence of a specific sequence and the lack of heparin and collagen binding domains of the vitronectin indicate that nectinepsin is a new extracellular matrix protein. Furthermore, genomic Southern blot studies suggest that nectinepsin and vitronectin are encoded by different genes. Western blot analysis with an anti-human vitronectin antiserum revealed, in addition to the 65- and 70-kDa vitronectin bands, an immunoreactive protein of about 54 kDa in all tissues containing nectinepsin mRNA. It seems likely that the form of vitronectin found in chick egg yolk plasma by Nagano et al. ((1992) J. Biol. Chem. 267, 24863-24870) is the protein that corresponds to the nectinepsin cDNA. This new protein could be an important molecule involved in the early steps of the development.

Amino Acid Sequence↗
PubMed2004

Assembly of exogenous fibronectin by fibronectin-null cells is dependent on the adhesive substrate.

The role of endogenously synthesized fibronectin (FN) in assembly was studied with cells lacking or expressing FN. Cells were cultured as homogeneous or mixed populations on surfaces coated with different matrix proteins. Compared with FN-expressing cells, FN-null cells poorly assembled exogenous plasma FN (pFN) when adhered to vitronectin or the recombinant cell-binding domain (III(7-10)) of FN. Vitronectin had a suppressive effect that was overcome by co-adsorbed pFN or laminin-1 but not by soluble FN. In co-cultures of FN-expressing cells and FN-null cells, endogenous FN was preferentially assembled around FN-expressing cells regardless of the adhesive ligand. If the adhesive ligand was vitronectin, exogenous pFN assembled preferentially around cells expressing cellular FN or recombinant EDa- or EDa+ FN. In co-cultures on vitronectin of FN-null cells and beta(1) integrin subunit-null cells, fibrils of cellular FN and pFN were preferentially deposited by FN-null (beta(1)-expressing) cells immediately adjacent to (FN-secreting) beta(1)-null cells. In co-cultures on vitronectin of FN-null cells and beta(1)-null cells expressing a chimera with the extracellular domain of beta(1) and the cytoplasmic domain of beta(3), preferential assembly was by the chimera-expressing cells. These results indicate that the adhesive ligand is a determinant of FN assembly by cells not secreting endogenous FN (suppressive if vitronectin, non-suppressive but non-supportive if III(7-10), supportive if pFN or laminin-1) and suggest that efficient interaction of freshly secreted cellular FN with a beta(1) integrin, presumably alpha(5)beta(1), substitutes for integrin-mediated adherence to a preformed matrix of laminin-1 or pFN to support assembly of FN.

Animals↗
PubMed1990

Isolation of plasminogen activator inhibitor-2 (PAI-2) from human placenta. Evidence for vitronectin/PAI-2 complexes in human placenta extract.

Plasminogen activator inhibitor-2 (PAI-2), found in human placenta and pregnancy plasma, was prepared in a highly purified and functionally active form from human placenta. The purification was achieved by a combination of Rivanol and ammonium sulfate precipitation, followed by chromatography on DEAE Affigel Blue, hydroxylapatite and phenylalanine-Sepharose. PAI-2, which is precipitated by low Rivanol concentrations, can be selectively redissolved from the pellet by increasing the Rivanol concentration in the presence of a reducing agent, i.e. dithiothreitol. The purified protein shows a molecular mass of 45 kDa in SDS PAGE, cross-reacts with monoclonal antibodies against PAI-2 (Mab'PAI-2), and inhibits the amidolytic activity of urokinase-type plasminogen activator (u-PA) towards the chromogenic substrate Glu-Gly-Arg-pNA (S-2444). The specific activity of the purified inhibitor was 52,300 units/mg, attaining 71,000 units/mg in peak fractions. In the immunopurification of placental extract on anti-PAI-2 Sepharose, the eluate showed the expected reaction with Mab' PAI-2, and it also cross-reacted with anti-vitronectin serum. In order to complement these results, anti-vitronectin Sepharose was used for immunopurification of placenta extract. In Western Blot experiments the eluates of anti PAI-2 Sepharose and anti-vitronectin Sepharose both showed a heterogeneous pattern of high molecular weight bands recognized by either polyclonal antiserum against vitronectin or Mab'PAI-2. In either case, reduction of the eluates releases mainly a 45-kDa band, which is recognized by Mab'PAI-2, or 80-kDa and 76-kDa bands recognized by anti-serum against vitronectin. These data suggest that the predominant form of PAI-2 in placenta extract is heterogeneous and of high molecular mass, containing complexes in which vitronectin is covalently bound to PAI-2 by disulfide bridges.

Blotting, Western↗
PubMed1991

Arginine-glycine-aspartic acid binding leading to molecular stabilization between integrin alpha v beta 3 and its ligand.

Cell adhesion is characterized by an integrin-mediated ligand binding event followed by reorganization of the actin-cytoskeleton leading to cell spreading and/or migration. In this report we examine the role of integrin alpha v beta 3 in mediating cell attachment to vitronectin or a RGD-containing peptide in the presence of cytochalasin B to prevent actin polymerization. Under these conditions cell attachment to a RGD-containing peptide can be dissociated by excess soluble ligand whereas cells attached to vitronectin cannot. These results suggest that alpha v beta 3-mediated cell attachment to vitronectin results in a highly stabilized interaction that is independent of the actin-cytoskeleton. To investigate the molecular nature of this interaction alpha v beta 3 was purified to homogeneity, and its binding properties toward various ligands were measured in a solid-phase receptor assay. The data indicate that alpha v beta 3 binds to vitronectin or fibronectin in a nondissociable manner whereas a RGD-containing peptide derived from vitronectin binds specifically but is completely dissociable with a Kd of 9.4 x 10(-7) M. Moreover, chemical modification of alpha v beta 3 with limited glutaraldehyde treatment allowed vitronectin to bind in a RGD-dependent and dissociable manner, suggesting that receptor conformational changes or specific amino acid residues proximal to the ligand binding site(s) are involved in the stabilization event. Thus, in the absence of cytoskeletal proteins or other cellular components, integrin alpha v beta 3-ligand binding involves recognition of the RGD sequence leading to a highly stabilized protein-protein association.

Amino Acid Sequence↗
PubMed1993

The alpha v beta 3 integrin associates with a 190-kDa protein that is phosphorylated on tyrosine in response to platelet-derived growth factor.

Integrins are cell surface heterodimers that mediate cell adhesion to the extracellular matrix. We show that in mouse 3T3 fibroblasts the alpha v beta 3 integrin (vitronectin receptor) coprecipitates with a tyrosine-phosphorylated 190-kDa protein, as detected by antibodies to phosphotyrosine. Three different antibodies to the vitronectin receptor, all of which precipitate the alpha/beta complex, coprecipitated a 190-kDa protein. The three antibodies were raised against the purified placental vitronectin receptor, the platelet alpha IIb beta 3 integrin, and the cytoplasmic domain of the alpha v subunit. The association was specific for the vitronectin receptor, since an antibody to the alpha 5 beta 1 integrin (fibronectin receptor) did not coprecipitate any tyrosine-phosphorylated protein. The phosphorylation of the 190-kDa protein was observed only following cell activation by platelet-derived growth factor, which is known to stimulate tyrosine kinase activity and to modulate cell adhesion. Antibodies raised against the platelet-derived growth factor alpha and beta receptors (M(r) = 170,000 and 190,000, respectively) did not recognize the 190-kDa, integrin-associated phosphorylated protein. Occupancy of the vitronectin receptor by one of its ligands, vitronectin, resulted in an increased amount of tyrosine phosphorylation of the 190-kDa protein. Our data suggest that the association of tyrosine-phosphorylated proteins with integrins may play an important role in growth factor-mediated modulation of cell adhesion.

3T3 Cells↗
PubMed1987

Influence of stereochemistry of the sequence Arg-Gly-Asp-Xaa on binding specificity in cell adhesion.

Peptides containing the tripeptide sequence Arg-Gly-Asp can duplicate or inhibit the cell attachment-promoting effects of fibronectin and vitronectin. Peptides analogous to a prototype peptide, Gly-Arg-Gly-Asp-Ser-Pro-Cys, the sequence of which was taken from the cell attachment site of fibronectin, were assayed for their relative abilities to inhibit the attachment of cells to a fibronectin or vitronectin substrate. A peptide having the L-Arg residue replaced with D-Arg showed no difference in this capacity, whereas substituting Gly with D-Ala or L-Asp with D-Asp resulted in completely inactive peptides. Replacement of L-Ser with D-Ser drastically reduced the influence that the resulting peptide had on the vitronectin interaction, but this peptide showed little difference in its effect on the binding of cells to fibronectin when compared with the prototype peptide. Furthermore, substitution of the Ser with L-Asn resulted in a peptide that had an apparent increased preference for the fibronectin receptor and decreased preference for the vitronectin receptor. Conversely, threonine in this position gave a peptide with increased preference for the vitronectin receptor, whereas L-Pro in this position gave a completely inactive peptide. Finally, by cyclicizing the prototype peptide to restrict its conformational flexibility, a peptide was obtained that was a much improved inhibitor of attachment of cells to vitronectin and yet nearly inactive with respect to the interactions of cells with fibronectin substrates. These studies lend support to the hypothesis that different Arg-Gly-Asp-directed adhesion receptors can recognize differences in the conformation and environment of the Arg-Gly-Asp tripeptide, and they establish the feasibility of obtaining synthetic probes that are more selective for individual receptors than are the peptides modeled after the natural sequences of adhesive extracellular matrix molecules.

Amino Acid Sequence↗
PubMed2001

alpha(v)beta(3) integrin engagement modulates cell adhesion, proliferation, and protease secretion in human lymphoid tumor cells.

OBJECTIVE: The mechanisms used by human lymphoproliferative diseases to invade locally and metastasize are thought to be similar to those developed by solid tumors, including cell proliferation and secretion of extracellular matrix-degrading enzymes following adhesion to extracellular matrix proteins. Hence, the ability of Namalwa (Burkitt's lymphoma), U266 (multiple myeloma), and CEM (T-cell lymphoblastic leukemia) cells to interact with the extracellular matrix components vitronectin and fibronectin was determined. Fresh bone marrow plasma cells from patients with multiple myeloma also were studied. MATERIALS AND METHODS: Engagement of alpha(v)beta(3) integrin, formation and protein composition of focal adhesion contacts on the cell surface, phosphorylation of several signal transduction proteins in the contacts, cell proliferation, and enzyme secretion were studied following adhesion to vitronectin and fibronectin. RESULTS: All three lines adhered to immobilized vitronectin and fibronectin. Adhesion was fully prevented by neutralizing monoclonal anti-alpha(v)beta(3) integrin antibody. Integrin engagement caused the formation of phosphorylated pp60(src)/focal adhesion kinase complexes and the aggregation of focal adhesion plaques containing the beta(3) integrin subunit, the cytoskeletal proteins vinculin, cortactin, and paxillin, the tyrosine kinases focal adhesion kinase and pp60(src), the adapter protein Grb-2, and the mitogen-activated protein kinase ERK-2. Free and immobilized vitronectin and fibronectin stimulated the proliferation of cells under serum-free conditions and the production and release of urokinase-type plasminogen activator, and increased the release of the activated forms of matrix metalloproteinase-2 and matrix metalloproteinase-9 in an alpha(v)beta(3) integrin-dependent manner. Similar results were obtained in myeloma plasma cells. CONCLUSIONS: The demonstrated ability of lymphoid tumor cells to interact with the extracellular matrix components vitronectin and fibronectin via alpha(v)beta(3) integrin can be interpreted as evidence of a novel mechanism for their invasion and spreading. This interaction allows them to adhere to the substratum and enhances their proliferation and protease secretion.

Antibodies, Monoclonal↗
PubMed1996

Integrin alphavbeta3 mediates chemotactic and haptotactic motility in human melanoma cells through different signaling pathways.

Distinctions between chemotaxis and haptotaxis of cells to extracellular matrix proteins have not been defined in terms of mechanisms or signaling pathways. Migration of A2058 human melanoma cells to soluble (chemotaxis) and substratum-bound (haptotaxis) vitronectin, mediated by alphav beta3, provided a system with which to address these questions. Both chemotaxis and haptotaxis were completely inhibited by treatment with RGD-containing peptides. Chemotaxis was abolished by a blocking antibody to alphavbeta3 (LM609), whereas haptotaxis was inhibited only by approximately 50%, suggesting involvement of multiple receptors and/or signaling pathways. However, blocking antibodies to alphavbeta5, also present on A2058 cells, did not inhibit. Pertussis toxin treatment of cells inhibited chemotaxis by >80%, but did not inhibit haptotaxis. Adhesion and spreading over vitronectin induced the phosphorylation of paxillin on tyrosine. In cells migrating over substratum-bound vitronectin, tyrosine phosphorylation of paxillin increased 5-fold between 45 min and 5 h. Dilutions of anti- alphavbeta3 that inhibited haptotaxis also inhibited phosphorylation of paxillin (by approximately 50%) and modestly reduced cell spreading. In contrast, soluble vitronectin (50-100 microg/ml) did not induce tyrosine phosphorylation of paxillin. The data suggest that soluble vitronectin stimulates chemotaxis predominantly through a G protein-mediated pathway that is functionally linked to alphavbeta3. Haptotaxis is analogous to directional cell spreading and requires alphavbeta3-mediated tyrosine phosphorylation of paxillin.

Amino Acid Sequence↗
PubMed1995

Ligation of endothelial alpha v beta 3 integrin increases capillary hydraulic conductivity of rat lung.

Complement-mediated pulmonary edema results from increases in lung capillary hydraulic conductivity (Lp), possibly by receptor-mediated mechanisms. We considered the Lp effects of vitronectin and the vitronectin-containing complement complex SC5b-9, which ligate the integrin alpha v beta 3. Vitronectin, SC5b-9, and SC5b-9-enriched zymosan-activated serum all rapidly increased Lp, as determined by the split-drop technique in single lung capillaries of rat lung. The Lp increases were inhibited by a monospecific (LM609) and a polyclonal (R838) antibody against the alpha v beta 3 integrin but not by an irrelevant monoclonal antibody isotype matched with LM609, by a monoclonal antibody against the alpha v beta 5 integrin, or by preimmune rabbit serum. Vitronectin monomers failed to increase Lp. The tyrosine kinase blockers genistein and methyl 2,5-dihydroxycinnamate caused significant concentration-dependent inhibitions of Lp increases due to vitronectin and zymosan-activated serum. By contrast, the protein kinase C blocker calphostin C had no major effect. We conclude that (1) multivalent ligation of the luminally located alpha v beta 3 integrin of lung capillary endothelium increases transcapillary liquid flux, and (2) the dominant signal transduction pathway for this effect occurs through tyrosine kinase activation.

Animals↗
PubMed1996

Serine 752 in the cytoplasmic domain of the beta 3 integrin subunit is not required for alpha v beta 3 postreceptor signaling events.

A naturally occurring point mutation (Ser752Pro substitution) in the beta subunit cytoplasmic domain of the platelet fibrinogen receptor GPIIb-IIIa (integrin alpha IIb beta 3), causing Glanzmann's thrombasthenia, has been shown to abrogate bidirectional transmembrane signaling of GPIIb-IIIa when expressed in heterologous cells (Chen YP, 1994, Blood 84, 1857-1865). As the vitronectin receptor alpha v beta 3 constitutively mediates cell attachment to RGD containing extracellular matrix proteins, the purpose of this study was to explore the regulatory role of Ser752 in alpha v beta 3 vitronectin receptor function, by cotransfecting recombinant human alpha v cDNA together with human beta 3 mutant cDNA into Chinese hamster ovary (CHO) cells. CHO cells expressing wild type human alpha v beta 3 acquired the ability to attach and spread on fibrinogen and von Willebrand factor, in contrast to non transfected CHO cells that only bound to vitronectin and fibronectin. Overexpression of a truncated recombinant beta 3 subunit (beta 3 delta 744) generated alpha v (hamster) beta 3 (human) chimers that mediated attachment but lost the ability to promote cell spreading on vitronectin, von Willebrand factor and fibrinogen, and to concentrate in focal contact sites, demonstrating a negative effect of beta 3 delta 744 on alpha v beta 3 dependent postreceptor occupancy events. Transfection of beta 3Ser752Pro reproduced the same negative effect as beta 3 delta 744, whereas beta 3Ser752Ala restored normal receptor function by allowing pronounced attachment and spreading on fibrinogen and von Willebrand factor. Our results provide evidence that (1) the C-terminal cytoplasmic domain of beta 3 (amino acids 744-762) is essential for alpha v beta 3 integrin postreceptor occupancy events; (2) within this domain, the Ser752Pro mutation affects alpha v beta 3 postreceptor occupancy events by preventing cell spreading and focal contact localization; (3) the defective receptor function of the vitronectin receptor alpha v beta 3 is due to the presence of Pro752, rather than the absence of Ser752, as a Ser to Ala substitution at position 752 restores normal beta 3 integrin cell spreading and adhesive plaque formation.

Alanine↗
PubMed1996

Modulation of alpha 5 beta 1 and alpha V beta 3 integrins on the cell surface during mitosis.

One of the hallmarks of cells undergoing mitotic division is their rounded morphology and reduced adhesion to the substratum. We have studied and compared the attachment of interphase and mitotic cells to substrata coated with fibronectin and vitronectin. We have found that adhesion of mitotic cells, as compared to interphase cells, is significantly reduced to fibronectin, but is higher to vitronectin. These results correlate well with the expression of alpha 5 beta 1 and alpha V beta 3 integrins, the respective receptors for fibronectin and vitronectin, on the cell surface. Mitotic cells show higher levels of alpha V beta 3 and very low levels of alpha 5 beta 1 proteins on the cell surface as compared to interphase cells. This difference in the levels of these integrins also reflects in the total amounts of fibronectin and vitronectin present on the cell surface of these cells. We have further shown, by flow cytometry, that binding of vitronectin, or the synthetic peptide -GRGDSP-, causes an increase in the intracellular levels of Ca2+ in mitotic cells, but no change is seen in the interphase cells. Binding of fibronectin to either of these cells fails to elicit any response. One interesting feature of our results is that the levels of total, i.e., cytoplasmic plus membrane bound, alpha 5 beta 1 and alpha V beta 3 integrins of mitotic and interphase cells remain the same, thus implying an alteration in the distribution of integrin chains between the plasma membrane and the cytoplasm during the conversion of interphase cells into the mitotic phase.

Animals↗
PubMed1989

Effect of inhibitors of N-linked oligosaccharide processing on the cell surface expression of a melanoma integrin.

The role of trimming and processing of N-linked oligosaccharides on the cell surface expression of the melanoma vitronectin receptor, a member of the integrin family of cell adhesion receptors, was examined by using specific glucosidase and mannosidase inhibitors. Inhibition of glucosidases I and II by castanospermine or N-methyldeoxynojirimycin delayed the vitronectin receptor alpha/beta chain heterodimer assembly and alpha chain cleavage and resulted in a decrease in the level of expression cell surface receptor. Conversely, the vitronectin receptor synthesized in the presence of the mannosidase I and II inhibitors, 1-deoxymannojirimycin and swainsonine, was transported normally to the cell surface with its alpha chain N-linked oligosaccharides in an endoglycosidase H-sensitive form. In the presence of swainsonine, time course studies of the cell surface replacement of control, endoglycosidase H-resistant receptor with an endoglycosidase H-sensitive form demonstrated a vitronectin receptor half-life of approximately 15-16 h. These studies provide evidence that the rates of assembly, proteolytic cleavage, and cell surface expression of the melanoma vitronectin receptor are dependent on the initial trimming of glucosyl residues from the alpha chain N-linked oligosaccharides.

Cell Line↗
PubMed1991

Characterization of integrin chains in normal and neoplastic human pancreas.

Integrins are a complex family of non-covalently linked heterodimeric glycoproteins which function as cell adhesion molecules, interacting with extracellular matrix molecules such as laminin, fibronectin, vitronectin, and collagen, and also having a role in intercellular adhesion. Each integrin subfamily is characterized by a common beta chain associated with variable alpha chains. We have examined, using immunohistological methods, the expression of the VLA (very late activation) family comprising beta 1 in association with alpha 1-6, and also alpha 6 in association with beta 4, the LFA beta chain beta 2, and the vitronectin receptor, in association with beta 1 or beta 5 and as the complex alpha v beta 3. Cryostat sections of normal pancreas, pancreatic adenocarcinomas, and ampullary tumours were studied together with six pancreatic carcinoma cell lines. Normal pancreas showed expression of beta 1 in all parenchyma. alpha 2 and alpha 6 had a similar distribution whereas alpha 3 expression was confined to ducts, including the very smallest radicles. Staining along the basement membranes of ducts was seen with beta 4 and the anti-vitronectin alpha v chain receptor antibody 13C2. Islet cells failed to stain with any antibody. No staining of epithelial components was seen with antibodies to alpha 1, alpha 4, alpha 5, or to the alpha v beta 3 form of the vitronectin receptor (beta 3 and alpha v beta 3 using the antibody 23C6). Pancreatic adenocarcinomas and ampullary tumours showed expression of alpha 2, alpha 3, alpha 6, beta 1, beta 4, and the vitronectin receptor (alpha v associated with beta 1 or beta 5).(ABSTRACT TRUNCATED AT 250 WORDS)

Basement Membrane↗
PubMed1993

Evidence for an extra-cellular function for protein kinase A.

In addition to its intra-cellular functions, cAMP-dependent protein kinase (PKA) may well have an extra-cellular regulatory role in blood. This suggestion is based on the following experimental findings: (a) Physiological stimulation of blood platelets brings about a specific release of PKA, together with its co-substrates ATP and Mg++; (b) In human serum, an endogenous phosphorylation of one protein (p75, M(r) 75 kDa) occurs; this phosphorylation is enhanced by addition of cAMP and blocked by the Walsh-Krebs specific PKA inhibitor; (c) No endogenous phosphorylation of p75 occurs in human plasma devoid of platelets, but the selective labeling of p75 can be reproduced by adding to plasma the pure catalytic subunit of PKA; (d) p75 was shown to be vitronectin (V), a multifunctional protein implicated in processes associated with platelet activation, and thus a protein whose function may require modulation for control; (e) The phosphorylation of vitronectin occurs at one site (Ser378) which, at physiological pH, is buried in its two-chain form (V65 + 10) but it becomes 'exposed' in the presence of glycosaminoglycans (GAGs) e.g. heparin or heparan sulfate. Such a transconformation may be used for targeting the PKA phosphorylation to vitronectin molecules bound to GAGs, for example in the extracellular matrix or on cell surfaces; (f) From the biochemical point of view (Km values and physiological concentrations) the phosphorylation of vitronectin can take place at the locus of a hemostatic event; (g) The phosphorylation of Ser378 in vitronectin alters its function, since it significantly reduces its ability to bind the inhibitor-1 of plasminogen activator(s) (PAI-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1998

Functional effects of single amino acid substitutions in the region of Phe113 to Asp138 in the plasminogen activator inhibitor 1 molecule.

Thirteen amino acid substitutions have been introduced within the stretch Phe113 to Asp138 in the plasminogen activator inhibitor 1 (PAI-1) molecule by site-directed mutagenesis. The different proteins and wild-type (wt) PAI-1 have been overexpressed in Escherichia coli and purified by chromatography on heparin-Sepharose and on anhydrotrypsin-agarose. The PAI-1 variants have been characterized by their reactivity with tissue plasminogen activator (tPA), interactions with vitronectin or heparin, and stability. Most PAI-1 variants, except for Asp125-->Lys, Phe126-->Ser and Arg133-->Asp, displayed a high spontaneous inhibitory activity towards tPA, which did not change greatly on reactivation with 4 M guanidinium chloride, followed by dialysis at pH 5.5. The variants Asp125-->Lys and Arg133-->Asp became much more active after reactivation and they were also more rapidly transformed to inactive forms (t12 22-31 min) at physiological pH and temperature than the other variants. However, in the presence of vitronectin they were both almost equally stable (t12 2.3 h) as wtPAI-1 (t12 3.0 h). The mutant Glu130-->Lys showed an increased stability, both in the absence and in the presence of vitronectin compared with wtPAI-1. Nevertheless a similar affinity between all the active PAI-1 variants and vitronectin was observed. Further, all mutants, including the three mutants with low activity, were to a large extent adsorbed on anhydrotrypsin-agarose and were eluted in a similar fashion. In accordance with these data, the three variants with a low activity were all to a large extent cleaved as a result of their reaction with tPA, suggesting that they occurred predominantly in the substrate conformation. Our results do not support the presence of a binding site for vitronectin in this part of the molecule, but rather that it might be involved in controlling the active PAI-1 to substrate transition. Partly, this region of the PAI-1 molecule (Arg115 to Arg118) seems also to be involved in the binding of heparin to PAI-1.

Amino Acid Substitution↗
PubMed1999

The role of plasminogen activator inhibitor-1 as inhibitor of platelet and megakaryoblastic cell adhesion.

In the present study the ability of plasminogen activator inhibitor type-1 (PAI-1) to interfere with platelet and megakaryoblastic cell adhesion was investigated. Both cell types exhibited integrin-dependent adhesion in a static system, mediated by alphaIIb beta3 on platelets and alpha v-integrins on different megakaryoblastic cell lines, even though they also expressed alphaIIb beta3. In a concentration-dependent manner, active, but not latent or complexed, PAI-1 abrogated cell adhesion onto vitronectin but not onto fibrinogen or other matrix substrata. Urokinase as well as thrombin neutralized the anti-adhesive effect of active PAI-1. The direct binding of vitronectin, but not of other matrix proteins, to integrin alphaIIb beta3 was blocked by active PAI-1 in a purified system. Since activated platelets release active and latent PAI-1 as well as structurally and functionally distinct forms of vitronectin, the described interactions appear to be physiologically significant. Co-distribution of vitronectin and PAI-1 at sites of fibrin polymers within platelet thrombi was demonstrated by transmission electron microscopy, suggesting an extracellular functional relationship of both release products with regard to cell adhesion. Our data emphasize the regulatory role of active PAI-1 in platelet adhesion to provisional matrix proteins as found during wound healing independent of its anti-proteolytic activity. Furthermore, megakaryocyte maturation may depend on the intact vitronectin-integrin adhesion system that is influenced by PAI-1, thereby proposing a regulatory role for the inhibitor in cellular differentiation.

Blood Platelets↗